Orofacial clefts (OFCs) of the lip and palate are common structural birth defects, affecting 1 in 700 newborns. These malformations cause significant morbidity, require extensive medical intervention, and pose serious individual, familial, and societal burdens. Children with OFCs undergo an average of six surgeries, have health care costs 800% greater than their peers, and have higher mortality rates at all stages of life. Prevention strategies for OFCs are elusive because our current understanding of causative factors is inadequate. Epidemiologic and traditional genetic studies have shown that OFCs are etiologically complex traits that likely result from gene-environment interactions. Epigenetic mechanisms are an exciting new focus in understanding the genesis of OFCs because they mediate the effect of environmental influences on the genome during sensitive embryonic periods. This application specifically focuses on DNA methylation because this epigenetic mechanism is environmentally sensitive and a practical target of prevention and therapeutic strategies. While implicated by multiple lines of evidence, the biological role of DNA methylation in orofacial development is not known. Moreover, while many molecular mediators of orofacial development have been identified, the specific genes and pathways that are regulated by DNA methylation during cleft pathogenesis have not been well defined. The studies proposed in this application utilize genetic and pharmacologic approaches to define the biological role of DNA methylation in orofacial development, and genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways that are sensitive to changes in methylation. Completion of these Aims will advance the field by dramatically increasing our understanding of the basic mechanisms by which DNA methylation regulates orofacial development, and how modulation of this key epigenetic process results in OFCs. The results are expected to link this environmentally-sensitive mechanism to a distinct biological process and cell population, and identify a focused group of epigenetically-regulated and environmentally-sensitive targets. This will provide a foundation for future focused research efforts that will advance our long term goal of developing prevention strategies for etiologically complex birth defects.